4.3 | Conjugation and release of β-Gal with/from droplets
Droplets with various DPPE-PEG-PyDTP concentrations were prepared as described above (see Supporting Information Table S1). After centrifugation, the precipitate containing the droplets in a microtube was suspended in β-Gal solution (1 μM in 1 mM MgCl2-PBS solution) and mechanically rotated in a refrigerator (4 °C) for 1 h. After centrifugation, the supernatant was removed, and the precipitate was resuspended in PBS. The process of centrifugation followed by the removal of supernatant and resuspension in PBS was repeated four times to remove the free β-Gal. For the reductive release assay, β-Gal-conjugated droplets were suspended in 200 μl of GSH solution (various concentrations from 0.010 mM to 10 mM in PBS) followed by incubation for 2 h. After centrifugation, the supernatant was recovered, desalted, freeze-dried, and rehydrated in 10 μl of 1.0 mM MgCl2-PBS solution. Subsequently, 4 μl of the solution was added to 116 μl of fluorogenic substrate solution (0.10 mM FDG, 1.0 mM MgCl2, and 112 mM ME in PBS), and then, the time-course of fluorescent intensity (Ex: 490 nm, Em: 514 nm) was assessed using a plate reader (CytationTM 5, Biotek Instruments, Vermont, USA). β-Gal concentration was determined based on the rate of increase in fluorescence.